Plasma high density lipoproteins (HDL) have a protective effect in atherosclerosis but the underlying mechanisms are incompletely understood. A part of the athero-protective effect of HDL may be related to its ability to reverse endothelial dysfunction, a characteristic feature of early atherosclerotic lesions involving decreased bioavailability of eNOS-derived NO and increased expression of cell adhesion molecules and inflammatory chemokines and cytokines. Many of the beneficial effects of HDL appear to be related to its ability to promote efflux of cholesterol and toxic oxysterols from cells via the ATP binding cassette transporters, ABCA1 and ABCG1. ABCG1 is highly expressed in endothelial cells and has an essential role in promoting efflux of cholesterol and 7- oxysterols to HDL. Our recent studies show that high cholesterol diet-fed Abcg1-/- develop a severe defect in eNOS-dependent relaxation of arterial segments, associated with accumulation of 7-ketocholesterol and disruption of the active, dimeric form of eNOS. In cholesterol-loaded human aortic endothelial cells (HAECs), knock-down of ABCG1 results in decreased eNOS activity and. This leads to the central hypothesis that ABCG1 has an anti-atherogenic role in endothelium. In Aim 1 we propose to investigate cellular mechanisms responsible for decreased eNOS activity and increased expression of cell adhesion molecules in ABCG1-deficient cells. The central concept we will test is whether there is increased formation of inhibitory eNOS/Caveolin complexes, increased caveolar localization and activation of signaling complexes involving NADPH oxidases. In Aims 2 and 3 we will use recently developed Abcg1flox/flox mice to carry out endothelial- specific knock-out of ABCG1. These mice will be crossed with Ldlr-/- mice to determine whether there is reduced eNOS activity, increased expression of cell adhesion molecules and increased atherogenesis. We will also determine if these mice are resistant to the athero-protective effects of increased HDL levels induced by infusions of reconstituted HDL or apoA-1 transgenesis. These studies will use novel animal and cellular models to provide new insights into the mechanisms of athero-protective effects of HDL involving arterial endothelium.